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Background: Microbiome research is providing important new insights into the metabolic interactions of complex microbial ecosystems involved in fields as diverse as the pathogenesis of human diseases, agriculture and climate change. Poor correlations typically observed between RNA and protein expression datasets make it hard to accurately infer microbial protein synthesis from metagenomic data. Additionally, mass spectrometry-based metaproteomic analyses typically rely on focused search sequence databases based on prior knowledge for protein identification that may not represent all the proteins present in a set of samples. Metagenomic 16S rRNA sequencing only targets the bacterial component, while whole genome sequencing is at best an indirect measure of expressed proteomes. Here we describe a novel approach, MetaNovo, that combines existing open-source software tools to perform scalable de novo sequence tag matching with a novel algorithm for probabilistic optimization of the entire UniProt knowledgebase to create tailored sequence databases for target-decoy searches directly at the proteome level, enabling metaproteomic analyses without prior expectation of sample composition or metagenomic data generation and compatible with standard downstream analysis pipelines. Results: We compared MetaNovo to published results from the MetaPro-IQ pipeline on 8 human mucosal-luminal interface samples, with comparable numbers of peptide and protein identifications, many shared peptide sequences and a similar bacterial taxonomic distribution compared to that found using a matched metagenome sequence database—but simultaneously identified many more non-bacterial peptides than the previous approaches. MetaNovo was also benchmarked on samples of known microbial composition against matched metagenomic and whole genomic sequence database workflows, yielding many more MS/MS identifications for the expected taxa, with improved taxonomic representation, while also highlighting previously described genome sequencing quality concerns for one of the organisms, and identifying an experimental sample contaminant without prior expectation. Conclusions: By estimating taxonomic and peptide level information directly on microbiome samples from tandem mass spectrometry data, MetaNovo enables the simultaneous identification of peptides from all domains of life in metaproteome samples, bypassing the need for curated sequence databases to search. We show that the MetaNovo approach to mass spectrometry metaproteomics is more accurate than current gold standard approaches of tailored or matched genomic sequence database searches, can identify sample contaminants without prior expectation and yields insights into previously unidentified metaproteomic signals, building on the potential for complex mass spectrometry metaproteomic data to speak for itself. Author summary: MetaNovo is an open-source software pipeline that integrates existing tools with a custom algorithm to produce targeted protein sequence databases for mass spectrometry metaproteomic analysis as an intermediate filtering step prior to standard sequence database search approaches. MetaNovo uses open-source tools to match peptide mass spectrometry spectra to sequence database entries in a parallelised and scalable manner and can be installed in a cluster or run standalone on a Linux machine. The software is scalable to the number of input files and search sequence database size. As inputs the software requires raw mass spectrometry data in MGF format, and a UniProt FASTA sequence database to search. The pipeline is relevant to users analysing protein data from multiple organisms, where the exact species composition is unknown, such as microbiome or environmental samples, and provides an avenue for analysis when matched metagenomics data or accurate taxonomic characterisation is not available as it infers the organisms and proteins present directly from the raw data and the parent sequence database. The targeted sequence database produced can be used with standard downstream peptide identification software that relies on a targeted input sequence database to search the raw data against and allows greater sensitivity in peptide spectral matching in metaproteomic datasets. [ABSTRACT FROM AUTHOR]

Background: The central role of proteins in diseases has made them increasingly attractive as therapeutic targets and indicators of cellular processes. Protein microarrays are emerging as an important means of characterising protein activity. Their accurate downstream analysis to produce biologically significant conclusions is largely dependent on proper pre-processing of extracted signal intensities. However, existing computational tools are not specifically tailored to the nature of these data and lack unanimity. Results: Here, we present the single-channel Protein Microarray Analysis Pipeline, a tailored computational tool for analysis of single-channel protein microarrays enabling biomarker identification, implemented in R, and as an interactive web application. We compared four existing background correction and normalization methods as well as three array filtering techniques, applied to four real datasets with two microarray designs, extracted using two software programs. The normexp, cyclic loess, and array weighting methods were most effective for background correction, normalization, and filtering respectively. Conclusions: Thus, here we provided a versatile and effective pre-processing and differential analysis workflow for single-channel protein microarray data in form of an R script and web application (https://metaomics.uct.ac.za/shinyapps/Pro-MAP/.) for those not well versed in the R programming language. [ABSTRACT FROM AUTHOR]

Karyopherin beta 1 (Kpnβ1) is the principal nuclear importer of cargo proteins and plays a role in many cellular processes. Its expression is upregulated in cancer and essential for cancer cell viability, thus the identification of its binding partners might help in the discovery of anti-cancer therapeutic targets and cancer biomarkers. Herein, we applied immunoprecipitation coupled to mass spectrometry (IP-MS) to identify Kpnβ1 binding partners in normal and cancer cells. IP-MS identified 100 potential Kpnβ1 binding partners in non-cancer hTERT-RPE1, 179 in HeLa cervical cancer, 147 in WHCO5 oesophageal cancer and 176 in KYSE30 oesophageal cancer cells, including expected and novel interaction partners. 38 binding proteins were identified in all cell lines, with the majority involved in RNA metabolism. 18 binding proteins were unique to the cancer cells, with many involved in protein translation. Western blot analysis validated the interaction of known and novel binding partners with Kpnβ1 and revealed enriched interactions between Kpnβ1 and select proteins in cancer cells, including proteins involved in cancer development, such as Kpnα2, Ran, CRM1, CCAR1 and FUBP1. Together, this study shows that Kpnβ1 interacts with numerous proteins, and its enhanced interaction with certain proteins in cancer cells likely contributes to the cancer state. [ABSTRACT FROM AUTHOR]

There is limited data on the role of asymptomatic STIs (aSTIs) on the risk of human immunodeficiency virus (HIV) acquisition in the male genital tract (MGT). The impact of foreskin removal on lowering HIV acquisition is well described, but molecular events leading to HIV acquisition are unclear. Here, in this pilot study, we show that asymptomatic urethral infection with Chlamydia trachomatis (CT) significantly impacts the foreskin proteome composition. We developed and optimized a shotgun liquid chromatography coupled tandem mass spectrometry (MS)-based proteomics approach and utilized this on foreskins collected at medical male circumcision (MMC) from 16 aSTI+ men and 10 age-matched STI-controls. We used a novel bioinformatic metaproteomic pipeline to detect differentially expressed (DE) proteins. Gene enrichment ontology analysis revealed proteins associated with inflammatory and immune activation function in both inner and outer foreskin from men with an aSTI. Neutrophil activation/degranulation and viral-evasion proteins were significantly enriched in foreskins from men with aSTI, whereas homotypic cell-cell adhesion proteins were enriched in foreskin tissue from men without an aSTI. Collectively, our data show that asymptomatic urethral sexually transmitted infections result in profound alterations in epithelial tissue that are associated with depletion of barrier integrity and immune activation. [ABSTRACT FROM AUTHOR]

A significant proportion of human immunodeficiency virus type 1 (HIV)-positive individuals are affected by the cognitive, motor and behavioral dysfunction that characterizes HIV-associated neurocognitive disorders (HAND). While the molecular etiology of HAND remains largely uncharacterized, HIV transactivator of transcription (HIV-Tat) is thought to be an important etiological cause. Here we have used mass spectrometry (MS)-based discovery proteomics to identify the quantitative, cell-wide changes that occur when non-transformed, differentiated human neurons are treated with HIV-Tat over time. We identified over 4000 protein groups (false discovery rate <0.01) in this system with 131, 118 and 45 protein groups differentially expressed at 6, 24 and 48 h post treatment, respectively. Alterations in the expression of proteins involved in gene expression and cytoskeletal maintenance were particularly evident. In tandem with proteomic evidence of cytoskeletal dysregulation we observed HIV-Tat induced functional alterations, including a reduction of neuronal intrinsic excitability as assessed by patch-clamp electrophysiology. Our findings may be relevant for understanding in vivo molecular mechanisms in HAND. [ABSTRACT FROM AUTHOR]

Biochemical evidence is vital for accurate genome annotation. The integration of experimental data collected at the proteome level using high resolution mass spectrometry allows for improvements in genome annotation by providing evidence for novel gene models, while validating or modifying others. Here, we report the results of a proteogenomic analysis of a reference strain of Mycobacterium smegmatis (mc²155), a fast growing model organism for the pathogenic Mycobacterium tuberculosis--the causative agent for Tuberculosis. By integrating high throughput LC/MS/MS proteomic data with genomic six frame translation and ab initio gene prediction databases, a total of 2887 ORFs were identified, including 2810 ORFs annotated to a Reference protein, and 63 ORFs not previously annotated to a Reference protein. Further, the translational start site (TSS) was validated for 558 Reference proteome gene models, while upstream translational evidence was identified for 81. In addition, N-terminus derived peptide identifications allowed for downstream TSS modification of a further 24 gene models. We validated the existence of six previously described interrupted coding sequences at the peptide level, and provide evidence for four novel frameshift positions. Analysis of peptide posterior error probability (PEP) scores indicates high-confidence novel peptide identifications and shows that the genome of M. smegmatis mc²155 is not yet fully annotated. Data are available via ProteomeXchange with identifier PXD003500. [ABSTRACT FROM AUTHOR]

Ser/Thr/Tyr protein phosphorylation plays a critical role in regulating mycobacterial growth and development. Understanding the mechanistic link between protein phosphorylation signaling network and mycobacterial growth rate requires a global view of the phosphorylation events taking place at a given time under defined conditions. In the present study we employed a phosphopeptide enrichment and high throughput mass spectrometry-based strategy to investigate and qualitatively compare the phosphoproteome of two mycobacterial model organisms: the fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG. Cells were harvested during exponential phase and our analysis detected a total of 185 phospho-sites in M. smegmatis, of which 106 were confidently localized [localization probability (LP) = 0.75; PEP = 0.01]. By contrast, in M. bovis BCG the phosphoproteome comprised 442 phospho-sites, of which 289 were confidently localized. The percentage distribution of Ser/Thr/Tyr phosphorylation was 39.47, 57.02, and 3.51% for M. smegmatis and 35, 61.6, and 3.1% for M. bovis BCG. Moreover, our study identified a number of conserved Ser/Thr phosphorylated sites and conserved Tyr phosphorylated sites across different mycobacterial species. Overall a qualitative comparison of the fast and slow growing mycobacteria suggests that the phosphoproteome of M. smegmatis is a simpler version of that of M. bovis BCG. In particular, M. bovis BCG exponential cells exhibited a much more complex and sophisticated protein phosphorylation network regulating important cellular cycle events such as cell wall biosynthesis, elongation, cell division including immediately response to stress. The differences in the two phosphoproteomes are discussed in light of different mycobacterial growth rates. [ABSTRACT FROM AUTHOR]

In the last 40 years only one new antitubercular drug has been approved, whilst resistance to current drugs, including rifampicin, is spreading. Here, we used the model organism Mycobacterium smegmatis to study mechanisms of phenotypic mycobacterial resistance, employing quantitative mass spectrometry-based proteomics to investigate the temporal effects of sub-lethal concentrations of rifampicin on the mycobacterial proteome at time-points corresponding to early response, onset of bacteriostasis and early recovery. Across 18 samples, a total of 3,218 proteins were identified from 31,846 distinct peptides averaging 16,250 identified peptides per sample. We found evidence that two component signal transduction systems (e.g. MprA/MprB) play a major role during initial mycobacterial adaptive responses to sub-lethal rifampicin and that, after dampening an initial SOS response, the bacteria supress the DevR (DosR) regulon and also upregulate their transcriptional and translational machineries. Furthermore, we found a co-ordinated dysregulation in haeme and mycobactin synthesis. Finally, gradual upregulation of the M. smegmatis-specific rifampin ADP-ribosyl transferase was observed which, together with upregulation of transcriptional and translational machinery, likely explains recovery of normal growth. Overall, our data indicates that in mycobacteria, sub-lethal rifampicin triggers a concerted phenotypic response that contrasts significantly with that observed at higher antimicrobial doses. [ABSTRACT FROM AUTHOR]